Purification and properties of 3 alpha-hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum from human intestine

3 alpha-Hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum, isolated from human intestinal bacteria, was capable of converting 3-ketoglycyrrhetic acid to 3 alpha-hydroxyglycyrrhetic acid. The enzyme was purified to homogeneity by means of butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A, Toyopearl HW-55S, and isoelectric focusing column chromatographies. The purified enzyme showed a specific activity of 156 mumol/min.mg toward 3 alpha-hydroxyglycyrrhetic acid, and showed a single band on SDS-polyacrylamide gel electrophoresis. The apparent molecular weight was 53,000, as estimated by gel filtration, and 30,000, as judged by SDS-polyacrylamide gel electrophoresis. Its isoelectric point was 5.2. The enzyme showed absolute specificity for the 3 alpha-hydroxyl and 3-ketonic groups of 18 alpha- or 18 beta-glycyrrhetic acid and required NADP+ and NADPH as cosubstrates. The enzyme did not act on any 3 alpha-hydroxyl or 3-ketonic group of steroids or bile acids. The enzyme is a novel type of enzyme, defined as 3 alpha-hydroxy-glycyrrhetinate dehydrogenase, being quite different from 3 alpha-hydroxysteroid dehydrogenase [EC 1.1.1.50].     

Aldose reductase inhibitors: flavonoids, alkaloids, acetophenones, benzophenones, and spirohydantoins of chroman

The inhibitory activity of various compounds, including 12 flavonoids, 10 alkaloids, 15 benzophenones, 5 acetophenones, and 7 spirohydantoins of chroman, was tested on rabbit lens aldose reductase, an enzyme involved in complications of diabetes. Almost all compounds tested were found to inhibit the enzyme at low concentrations (10(-5) M). The most potent inhibitor was 2R,4S-6-chloro-2-methylspiro(chroman-4,4'-imidazo-lidine+ ++)-2',5'-dione with an I50 value of 4.7 x 10(-8) M; other spirohydantoins showed similar potency. Polyhydroxybenzophenones were also potent inhibitors with an I50 value of about 10(-7) M. The possible structure-inhibitory activity relationships of the compounds tested are discussed.     

Time Course of Optical Quality and Intraocular Scattering after Refractive Lenticule Extraction

Purpose

To assess the time course of optical quality and intraocular scattering in relation to visual acuity after femtosecond lenticule extraction (FLEx) for the correction of myopia.

Methods

This study evaluated 36 eyes of 36 patients with spherical equivalents of −4.38±1.53 D [mean ± standard deviation] who underwent FLEx. Before surgery, and 1 week and 1, 3 and 6 months after surgery, we assessed the modulation transfer function (MTF) cutoff frequency, Strehl ratio, objective scattering index (OSI), and OQAS values (OVs), using a double-pass instrument. We also investigated the relationship of the OSI with corrected distance visual acuity (CDVA) preoperatively and postoperatively.

Results

The mean changes in MTF cutoff frequency, Strehl ratio, OSI, OV100%, OV20%, and OV9% preoperatively and 6 months postoperatively were −5.51±15.01, −0.03±0.07, 0.35±0.83, −0.17±0.48, −0.14±0.38, and −0.09±0.22, respectively. We found no significant preoperative correlation between the OSI and logMAR CDVA (Spearman rank correlation coefficient r = 0.068, p = 0.69), and modest, but significant correlations 1 week and 1, 3, and 6 months postoperatively (r = 0.572, r = 0.562, r = 0.542, r = 0.540, p<0.001, respectively).

Conclusions

FLEx induced a transient decrease in optical quality in association with an increase in intraocular scattering in the early postoperative period, possibly due to mild interface haze formation, but gradually recovered with time. It is suggested that this transient degradation in optical quality related to an increase in the intraocular scattering may result in a slight delay of CDVA recovery in the early postoperative period.     

Enzymatic Synthesis of the Crithidia Factors in Cell-free Extracts of Serratia indica

The concomitant production of formic acid and pterin compounds from guanosine-5′-triphosphate (GTP) has been found in cell-free extracts of Serratia indica. Among the pterin compounds, l-threo-neopterin–the major Crithidia factor in S. indica–, a cyclic phosphate of neopterin (cNP), d-erythro-neopterin and 6-hydroxymethyl pterin were detected and isolated. Formate-¹⁴C elimination from GTP-8-¹⁴C was quantitatively distributed in the ethyl acetate layer in the ehyl acetate-hydrochloric acid partition system. Carbon 8 of GTP was released as formic acid. Enzymatic production of formate and cNP was linear for 2 hr at 37°C. Formate production was proportional to the enzyme concentration. The optimum pH for formate elimination was observed around pH 8.6. Optimum temperature for the production of formate and cNP was 50°C. The apparent Km value of GTP for formate production was 6.2×10^?bm. Formate eliminating activity was activated by disodium phosphate but was inhibited by Mg²⁺ or AMP. Incorporation of GTP-U-¹⁴C into pterin compounds was also regulated with disodium phosphate. Effective incorporation into cNP and d-erythro-neopterin occurred in the presence of phosphate. When phosphate was omitted from the system, however, effective incorporation into 6-hydroxymethyl pterin was observed. The biosynthetic process of the Crithidia factors, i.e. l-threo-neopterin and cNP, from GTP in S. indica is also discussed.     

Formation of Pungent Principles in Fruits of Sweet Pepper,Capsicum annuum L. var. grossum during Post-harvest Ripening under Continuous Light

Pungent principles (Capsaicinoid(s)) were found to be produced in fruits of sweet pepper, Capsicum annuum L. var. grossum, during post-harvest ripening under continuous light. The initial formation was observed after 4 days’ ripening. After 7 days’ ripening, the capsaicinoids content in placenta increased to 12.9 μg per fruit, which was 2.5-fold of that in pericarp. No pungent principles were detected in fruits during ripening in the dark and in seeds under continuous light. In placenta, the formation of dihydrocapsaicin and nordihydrocapsaicin which are the vanillylamides of saturated branched fatty acids was higher than that of capsaicin which is the vanillylamide of an unsaturated one. Remarkable formation and accumulation of carotenoid were also observed during post-harvest ripening under continuous light.     

Aspartate kinase-independent lysine synthesis in an extremely thermophilic bacterium, Thermus thermophilus: lysine is synthesized via alpha-aminoadipic acid not via diaminopimelic acid

An aspartate kinase-deficient mutant of Thermus thermophilus, AK001, was constructed. The mutant strain did not grow in a minimal medium, suggesting that T. thermophilus contains a single aspartate kinase. Growth of the mutant strain was restored by addition of both threonine and methionine, while addition of lysine had no detectable effect on growth. To further elucidate the lysine biosynthetic pathway in T. thermophilus, lysine auxotrophic mutants of T. thermophilus were obtained by chemical mutagenesis. For all lysine auxotrophic mutants, growth in a minimal medium was not restored by addition of diaminopimelic acid, whereas growth of two mutants was restored by addition of alpha-aminoadipic acid, a precursor of lysine in biosynthetic pathways of yeast and fungi. A BamHI fragment of 4.34 kb which complemented the lysine auxotrophy of a mutant was cloned. Determination of the nucleotide sequence suggested the presence of homoaconitate hydratase genes, termed hacA and hacB, which could encode large and small subunits of homoaconitate hydratase, in the cloned fragment. Disruption of the chromosomal copy of hacA yielded mutants showing lysine auxotrophy which was restored by addition of alpha-aminoadipic acid or alpha-ketoadipic acid. All of these results indicated that in T. thermophilus, lysine was not synthesized via the diaminopimelic acid pathway, believed to be common to all bacteria, but via a pathway using alpha-aminoadipic acid as a biosynthetic intermediate.     

Evolution by polyploidy and gene regulation in Anura

The evolution of the metazoa has been characterized by gene redundancy, generated by polyploidy, tandem duplication and retrotransposition. Polyploidy can be detected by looking for duplicated chromosomes or segments of orthologous chromosomes in post-polyploid animals. It has been proposed that the evolutionary role of polyploidy is to provide extra-copies of genes, whose subsequent alteration leads to new functions, increased biological complexity, and, ultimately, speciation. We review the theory of evolution by genome duplication, basing our arguments on findings from autopolyploid anurans and fish, undergoing post-polyploidy diploidization. We conclude that: 1) the high genetic variability of autotetraploid anurans is a result of tetrasomic expression, based on studies of isozymes and other proteins. 2) Epigenetic mechanisms mediate the reduced expression or silencing of redundant copies of genes in the regulation of gene expression of these tetraploids. This conclusion is based on data concerning ribosomal and hemoglobin gene activity. 3) Duplication of the genome may have occurred more than once in the phylogeny of the anurans, as exemplified by 4n and 8n Leptodactylidae species.     

Characterization of a lysK gene as an argE homolog in Thermus thermophilus HB27

We conducted a chromosome walk to obtain a DNA fragment downstream of lysJ and found an argE homolog in a putative operon composed of lysJ-orfC-orfD-argE homologs. A knockout mutant of the argE homolog showed significantly slow growth on a minimal medium, and the growth was markedly improved by addition of lysine. We therefore termed this gene lysK. Purified LysK protein has deacetylating activities for both N(2)-acetyllysine and N(2)-acetylornithine at almost equal efficiency. These results suggest that lysK which may share an ancestor with argE functions not only for the lysine biosynthesis, but also for arginine biosynthesis in Thermus thermophilus.     

Synthesis and characterization of novel phenylindoles as potential probes for imaging of β-amyloid plaques in the brain

We synthesized a novel series of phenylindole (PI) derivatives and evaluated their biological activities as probes for imaging Aβ plaques in vivo. The affinity for Aβ plaques was assessed by an in vitro-binding assay using pre-formed synthetic Aβ aggregates. 2-Phenyl-1H-indole (2-PI) derivatives showed high affinity for Aβ42 aggregates with K_i values ranging from 4 to 32 nM. 2-PI derivatives clearly stained Aβ plaques in an animal model of AD. In biodistribution experiments using normal mice, 2-PI derivatives displayed sufficient uptake for imaging, ranging from 1.1% to 2.6% ID/g. Although additional modifications are necessary to improve uptake by and clearance from the brain, 2-PI derivatives may be useful as a backbone structure to develop novel Aβ imaging agents.     

Comparison of Astigmatic Correction after Femtosecond Lenticule Extraction and Small-Incision Lenticule Extraction for Myopic Astigmatism

Purpose

To compare postoperative astigmatic correction between femtosecond lenticule extraction (FLEx) and small-incision lenticule extraction (SMILE) in eyes with myopic astigmatism.

Methods

We examined 26 eyes of 26 patients undergoing FLEx and 26 eyes of 26 patients undergoing SMILE to correct myopic astigmatism (manifest astigmatism of 1 diopter (D) or more). Visual acuity, cylindrical refraction, the predictability of the astigmatic correction, and the astigmatic vector components using Alpin’s method, were compared between the two groups 3 months postoperatively.

Results

We found no statistically significant difference in manifest cylindrical refraction (p=0.74) or in the percentage of eyes within ± 0.50 D of their refraction (p=0.47) after the two surgical procedures. Moreover, no statistically significant difference was detected between the groups in astigmatic vector components, namely, surgically induced astigmatism (0.80), target induced astigmatism (p=0.87), astigmatic correction index (p=0.77), angle of error (p=0.24), difference vector (p=0.76), index of success (p=0.91), flattening effect (p=0.79), and flattening index (p=0.84).

Conclusions

Both FLEx and SMILE procedures are essentially equivalent in correcting myopic astigmatism using vector analysis, suggesting that the lifting or non-lifting of the flap does not significantly affect astigmatic outcomes after these surgical procedures.